AAHS ASPN ASRM - 2013 Annual Meeting - American Association ...

Nerve Fiber and Motor Neuron Count Variation with Time After Nerve Injury
Institution where the work was prepared: Washington University, Saint Louis, MO, USA
Ida K. Fox, MD; Daniel A. Hunter; Susan E. Mackinnon; Washington University College of Medicine
Purpose:
The regeneration and subsequent remodeling of peripheral nerves remains a poorly understood process. After transection and repair or interposition grafting,
the distal number of nerve fibers has been shown to change with time. The goal of this study was to assess both changes in the number of nerve fibers distal
to the repair and the corresponding number of retrograde labeled motor neuron cell bodies detected
Methods:
Lewis rat sciatic nerve underwent either transection and repair or interposition grafting of isologous sciatic nerve. Data was collected at 1, 3, 6, 9, 12 and 24
months post-procedure. To assess histomorphometric differences, peripheral nerve tissue distal to the repair or distal to the graft was collected. To quantify
motor neuron regeneration, retrograde tracer was applied distal to repair site or interposed graft. Lumbar spinal cord ventral horn tissue was collected and the
number of retrograde-labeled cell bodies in the ventral horn was counted.
Results:
Preliminary histomorphometric data shows augmentation then diminution in the number of distal nerve fibers with time. Overall, the transection and repair groups
have more numerous distal fibers compared to the grafted group—this held true for all times points. The number of retrograde labeled motor neuron cell bodies
showed a similar pattern of increase then decrease, however, the total numbers did not vary significantly between the two groups at the later times points.
Conclusion:
In studying nerve regeneration in the rodent model, the timing of outcomes assessment and nerve harvest is crucial. There is much to be learned about the
regeneration of peripheral nerves, especially in regard to motor regeneration. This work serve as the basis for further investigation of motor regeneration distal
to transection or graft interposition-requiring injury.
Use of Skin-Derived Stem Cells to Promote Peripheral Nerve Regeneration and Recovery from Chronic
Denervation
Institution where the work was prepared: University of Calgary, Hotchkiss Brain Institute, Calgary, AB, Canada
Sarah K. Walsh, BSc1; J. Biernaskie2; F. Miller2; Raj Midha, MD, MSc1; (1)University of Calgary, (2)University of Toronto
Easily accessible sources for stem cell transplantation from skin dermis (termed skin-derived precursors, or SKPs) have been isolated from embryonic and adult
murine skin (Toma et al., Nature Cell Biol, 2001) and have the ability to differentiate in vitro to neural crest cell types, including those with characteristics of
peripheral neurons and Schwann cells. Our recent work (McKenzie et al., J. Neurosci., 2006) showed that naïve SKPs or those differentiated toward a Schwann
cell-like phenotype (SKP-SCs) were able to survive and indeed myelinate regenerating axons in the crush-injured mouse sciatic nerve. In our ongoing study, we
are exploring the ability of SKPs to remain viable and differentiate within a chronically denervated nerve in order to ascertain their role in promoting nerve
regeneration. To this end, the sciatic nerves of CD-1 mice were transected and capped for 8 weeks to prevent reinnervation of the distal stump, creating a situation
of chronic denervation. Nerves were then repaired and SKPs or SKP-SCs (generated from GFP +ve mice according to Toma et al., 2001) were injected
into the subepineurium distal to the transection. Immunohistochemistry and confocal microscopy 4-12 weeks following transplantation is expected to reveal
survival and differentiation of both naïve and differentiated SKPs, represented as co-localization of GFP fluorescence and Schwann cell makers. Additionally,
we expect to observe improved regeneration outcomes when compared to diluent controls. This study also examined the possibility of seeding synthetic guidance
chambers with SKPs as a method of delivering a source of Schwann cells to nerve gaps often found in chronic denervation. Preliminary work in vitro
shows that SKPs cultured within the lumen of chitosan tubes attach evenly to the walls and display morphology and protein expression consistent with that
of Schwann cells. When SKP-seeded tubes are used to bridge a 5 mm gap in the mouse sciatic nerve, we expect that axonal regeneration through the tubes
will be comparable to Schwann cell-seeded tubes, and significantly improved over empty conduits alone. We therefore conclude that SKPs represent an accessible,
autologous source of stem cells for transplantation therapies that have potential to myelinate regenerating axons and improve regeneration outcomes in
a chronic nerve denervation scenario.
Collagen Nerve Protectors in Rat Sciatic Nerve Repair: A Functional and Mechanical Analysis
Institution where the work was prepared: Columbia University Medical Center, Department of Ortho. Surgery, New York, NY, USA
Austin G. Hayes, BS; Charles M. Jobin, MD; Yelena Akelina, DVM; Melvin P. Rosenwasser, MD; Columbia University Medical Center
Purpose:
Peripheral nerve repair is often complicated by connective tissue proliferation, formation of perineural adhesions, and inhibition of gliding with subsequent
nerve dysfunction. The use of bio-absorbable protective wraps may improve the functional outcomes of these repairs by inhibiting adhesions. Perineural scar
and nerve tethering can be mechanically tested by stretching devices. This study analyzed the motor and sensory recovery and mechanical stiffness in transected
rat sciatic nerves repaired with and without tubular collagen nerve protectors.
Methods:
Thirty Sprague-Dawley rats underwent unilateral sharp sciatic nerve transection and repair with four epineurial sutures and were randomly treated with or without
a circumferential collagen nerve protector, NeuraWrapTM. After ten weeks of healing, in vivo motor and sensory recovery was tested with extensor postural
thrust, latency of limb withdrawal from noxious (56C hotplate) stimuli, and walking track analysis with calculation of sciatic functional index (SFI). Rats were
then sacrificed and their sciatic nerves were stretched in situ with an Instron microtesting device at a rate of 20mm/min until failure. Force distraction curves
were plotted and the stiffness and maximum load were calculated. As a final measure of muscle reinnervation, the percentage of gastrocnemius muscle weight
lost between repaired and unoperated limbs was compared.
Results:
Of the thirty rats, two were sacrificed prior to testing, one due to infection (unwrapped), while another was lost during housing (wrapped). Significant differences existed
between repaired and uninjured nerves in nearly all measures. Functional recovery of repaired nerves was not significantly different between non-wrapped and
wrapped nerves as measured by percentage extensor thrust deficit (82%, 86%), limb withdrawal time (4.2sec, 3.5sec), SFI (-57, -60), or percentage of gastrocnemius
weight lost (45%, 47%). Mechanical testing also found no significant differences between non-wrapped and wrapped nerves in stiffness (0.55+/-0.14N-mm, 0.49+/-
0.13N-mm) and maximum load (2.97N, 2.70N). Repaired nerves, regardless of wrapping, were 1.4-fold stiffer than uninjured nerves (0.37+/-0.13N-mm) (p